In-depth evaluation of Gly-Sar transport parameters as a function of culture time in the Caco-2 cell model.

The aim of the present study was to investigate the influence of culture time on hPEPT1-mediated transport in Caco-2 cell monolayers. Peptide transport activity in Caco-2 cells grown in standard media and in a "rapid" 4-day model was first compared. The rapid 4-day Caco-2 cell model, cultured using a cocktail of growth factors and agonists, displayed lower peptide uptake capacity than Caco-2 cells grown for 4 days in conventional media, and was judged to be unsuitable for peptide transport studies. Peptide transport activity as well as monolayer integrity and tissue morphology were evaluated in the standard >21 days model as a function of the culture time. Peptide transport activity was studied using [14C]-glycylsarcosine ([14C]-Gly-Sar). Monolayer integrity was evaluated by transepithelial electrical resistance (TEER) measurements and [3H]-mannitol permeabilities. Tissue morphology and hPEPT1 expression were studied using confocal laser scanning microscopy (CLSM) and conventional staining/immunostaining. Caco-2 cells grown in conventional media became confluent after 3-4 days. Mannitol permeability decreased from day 5 to 21 and TEER increased steadily until approximately day 21. Apical hPEPT1 uptake activity appeared to be maximal in cells cultured for >21 days, whereas basolateral uptake reached a maximum already after 12 days in culture. In some of the passages studied, a secondary increase in hPEPT1 transport activity was observed in cells grown for >25 days. A large carrier-mediated transepithelial peptide flux component was evident from day 14.

[1]  V. Ganapathy,et al.  Inhibition of the H+/peptide cotransporter in the human intestinal cell line Caco-2 by cyclic AMP. , 1996, Biochemical and biophysical research communications.

[2]  I. Alferiev,et al.  A peptide prodrug approach for improving bisphosphonate oral absorption. , 2000, Journal of medicinal chemistry.

[3]  B. Rothen‐Rutishauser,et al.  Formation of Multilayers in the Caco-2 Cell Culture Model: A Confocal Laser Scanning Microscopy Study , 2000, Pharmaceutical Research.

[4]  M. Hediger,et al.  Differential Recognition of ACE Inhibitors in Xenopus Laevis Oocytes Expressing Rat PEPT1 and PEPT2 , 2000, Pharmaceutical Research.

[5]  C. Tse,et al.  Nucleoside transport in human colonic epithelial cell lines: evidence for two Na+-independent transport systems in T84 and Caco-2 cells. , 1999, Biochimica et biophysica acta.

[6]  T. Tsuruo,et al.  Functional expression of P-glycoprotein in apical membranes of human intestinal Caco-2 cells. Kinetics of vinblastine secretion and interaction with modulators. , 1993, The Journal of biological chemistry.

[7]  A. Blais,et al.  Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon , 2005, The Journal of Membrane Biology.

[8]  S. Frokjaer,et al.  Dipeptide model prodrugs for the intestinal oligopeptide transporter. Affinity for and transport via hPepT1 in the human intestinal Caco-2 cell line. , 2001, Journal of controlled release : official journal of the Controlled Release Society.

[9]  V. Ganapathy,et al.  Valacyclovir: a substrate for the intestinal and renal peptide transporters PEPT1 and PEPT2. , 1998, Biochemical and biophysical research communications.

[10]  G. Amidon,et al.  Intestinal absorption mechanism of dipeptide angiotensin converting enzyme inhibitors of the lysyl-proline type: lisinopril and SQ 29,852. , 1989, Journal of pharmaceutical sciences.

[11]  G. Amidon,et al.  Cellular Uptake Mechanism of Amino Acid Ester Prodrugs in Caco-2/hPEPT1 Cells Overexpressing a Human Peptide Transporter , 1998, Pharmaceutical Research.

[12]  Hongshi Yu,et al.  Evidence for Diminished Functional Expression of Intestinal Transporters in Caco-2 Cell Monolayers at High Passages , 1997, Pharmaceutical Research.

[13]  Keisuke Konishi,et al.  New and better protocols for a short-term Caco-2 cell culture system. , 2002, Journal of pharmaceutical sciences.

[14]  H. Saito,et al.  Dipeptide transporters in apical and basolateral membranes of the human intestinal cell line Caco-2. , 1993, The American journal of physiology.

[15]  S. Frokjaer,et al.  Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line. , 2001, American journal of physiology. Gastrointestinal and liver physiology.

[16]  J. Beaulieu,et al.  Transient mosaic patterns of morphological and functional differentiation in the Caco-2 cell line. , 1992, Gastroenterology.

[17]  É. Brot-Laroche,et al.  Expression and localization of GLUT-5 in Caco-2 cells, human small intestine, and colon. , 1992, The American journal of physiology.

[18]  J. Moberly,et al.  Transepithelial transport of cholyltaurine by Caco-2 cell monolayers is sodium dependent. , 1993, American Journal of Physiology.

[19]  V. Ganapathy,et al.  Transport of valganciclovir, a ganciclovir prodrug, via peptide transporters PEPT1 and PEPT2. , 2000, Journal of pharmaceutical sciences.

[20]  S. Frokjaer,et al.  Epidermal growth factor and insulin short-term increase hPepT1-mediated glycylsarcosine uptake in Caco-2 cells. , 2003, Acta physiologica Scandinavica.

[21]  W. Kramer,et al.  Interaction of renin inhibitors with the intestinal uptake system for oligopeptides and beta-lactam antibiotics. , 1990, Biochimica et biophysica acta.

[22]  S. Adibi,et al.  Characterization of an oligopeptide transporter in renal lysosomes. , 2000, Biochimica et biophysica acta.

[23]  D. Thwaites,et al.  Substrate specificity of the di/tripeptide transporter in human intestinal epithelia (Caco‐2): identification of substrates that undergo H+‐coupled absorption , 1994, British journal of pharmacology.

[24]  J. Finley,et al.  The Influence of Culture Time and Passage Number on the Morphological and Physiological Development of Caco-2 Cells , 1997, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[25]  A. Guo,et al.  Direct evidence for peptide transporter (PepT1)-mediated uptake of a nonpeptide prodrug, valacyclovir. , 1998, Biochemical and biophysical research communications.

[26]  D. Thwaites,et al.  Na(+)-independent, H(+)-coupled transepithelial beta-alanine absorption by human intestinal Caco-2 cell monolayers. , 1993, The Journal of biological chemistry.

[27]  R Hori,et al.  H+ coupled active transport of bestatin via the dipeptide transport system in rabbit intestinal brush-border membranes. , 1992, The Journal of pharmacology and experimental therapeutics.

[28]  F. Hirche,et al.  Decisive structural determinants for the interaction of proline derivatives with the intestinal H+/peptide symporter. , 1999, European journal of biochemistry.

[29]  R. Neubert,et al.  Intestinal Transport of β-Lactam Antibiotics: Analysis of the Affinity at the H+/Peptide Symporter (PEPT1), the Uptake into Caco-2 Cell Monolayers and the Transepithelial Flux , 2004, Pharmaceutical Research.

[30]  H. Saito,et al.  Functional characteristics of basolateral peptide transporter in the human intestinal cell line Caco-2. , 1999, American journal of physiology. Gastrointestinal and liver physiology.

[31]  B. H. Stewart,et al.  Transport properties are not altered across Caco-2 cells with heightened TEER despite underlying physiological and ultrastructural changes. , 1996, Journal of pharmaceutical sciences.

[32]  I. Hidalgo,et al.  Transport of bile acids in a human intestinal epithelial cell line, Caco-2. , 1990, Biochimica et biophysica acta.

[33]  H. Saito,et al.  Transepithelial transport of oral cephalosporins by monolayers of intestinal epithelial cell line Caco-2: specific transport systems in apical and basolateral membranes. , 1992, The Journal of pharmacology and experimental therapeutics.

[34]  S. Adibi,et al.  Hormonal regulation of oligopeptide transporter Pept-1 in a human intestinal cell line. , 1999, American journal of physiology. Cell physiology.

[35]  S. Frokjaer,et al.  Prodrugs of purine and pyrimidine analogues for the intestinal di/tri-peptide transporter PepT1: affinity for hPepT1 in Caco-2 cells, drug release in aqueous media and in vitro metabolism. , 2003, Journal of controlled release : official journal of the Controlled Release Society.

[36]  Thomas J. Raub,et al.  Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. , 1989, Gastroenterology.

[37]  G. Amidon,et al.  Passive and Carrier-Mediated Intestinal Absorption Components of Two Angiotensin Converting Enzyme (ACE) Inhibitor Prodrugs in Rats: Enalapril and Fosinopril , 1989, Pharmaceutical Research.

[38]  H. Takeda,et al.  Effect of Lipopolysaccharide on Peptide Transporter 1 Expression in Rat Small Intestine and Its Attenuation by Dexamethasone , 2002, Digestion.

[39]  V Ganapathy,et al.  Improvement of L-dopa absorption by dipeptidyl derivation, utilizing peptide transporter PepT1. , 1998, Journal of pharmaceutical sciences.

[40]  Claude Roques,et al.  Correlation Between Oral Drug Absorption in Humans, and Apparent Drug Permeability in TC-7 Cells, A Human Epithelial Intestinal Cell Line: Comparison with the Parental Caco-2 Cell Line , 1998, Pharmaceutical Research.

[41]  M. Hediger,et al.  Human Intestinal H+/Peptide Cotransporter , 1995, The Journal of Biological Chemistry.

[42]  J. Polli,et al.  Development of a more rapid, reduced serum culture system for Caco-2 monolayers and application to the biopharmaceutics classification system. , 2000, International journal of pharmaceutics.

[43]  T. Kissel,et al.  Transepithelial Transport Properties of Peptidomimetic Thrombin Inhibitors in Monolayers of a Human Intestinal Cell Line (Caco-2) and Their Correlation to in Vivo Data , 1995, Pharmaceutical Research.